In previous work supported by this grant, we discovered a new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) that involves the intermediate phosphatidylinositol-5-phosphate (PI-5-P) and a family of enzymes called Type II PI5P4Ks. While the canonical pathway for PI-4,5- P2 involves the intermediate phosphatidylinositol-4-phosphate (PI-4-P) and results in production of PI-4,5- P2 at the plasma membrane, recent studies indicate that the PI-5-P/Type II PI5P4K pathway produces PI-4,5- P2 at intracellular membranes, including the endoplasmic reticulum. We have recently found that the Type II PISP4K enzymes are in a complex with a protein that binds to and regulates the insositol-l,4,5-trisphosphate (IP3) gated calcium channel of the endoplasmic reticulum. These results have led us to propose a model in which production of PI-4,5- P2 at the endoplasmic reticulum via the PI-5-P/Type II PI5P4K pathway provides local regulation of IP3 dependent calcium release from the endoplasmic reticulum. To determine the physiological relevance of the PI-5-P pathway, we generated mice lacking specific isoforms of Type II PI5P 4-kinases (Type IIbeta and Type IIalpha) and discovered that the Type IIbeta PI5P4K-/- mice have increased insulin sensitivity compared to their wild type littermates and also have reduced adiposity. We postulate that these results could be explained by a model in which disregulation of calcium release from internal pools influences insulin responses, including glucose uptake into skeletal muscle. Based on this model, we propose to 1) investigate the effect of deleting Type IIbeta and Type IIalpha PI5P4Ks on calcium release from intracellular stores; 2) investigate the possibility that the increased insulin sensitivity in muscle from Type IIbeta PI5P4K-/- mice results from disregulation of calcium release from intracellular stores; 3) investigate the effect of loss of Type IIa PI5P4K on calcium signaling pathways in hematopoietic cells where this enzyme is highly expressed and 4) identify new proteins that interact with PI-5-P or PI-4,5- P2 and which may play a role in this pathway. These studies suggest a new and unexpected mechanism for regulating cytosolic calcium and also suggest a new target for treatment of diabetes and obesity.